PROJECT SUMMARY Sensory hair cells within the inner ear are susceptible to damage and death from environmental toxins, including exposure to loud noise and some types of drugs. Because adult mammals have little to no capacity to regenerate hair cells, hair cell loss causes permanent hearing and balance impairments in humans. In contrast, nonmammalian vertebrates like fish, frogs, and birds can robustly regenerate hair cells throughout life, enabling functional recovery after damage in adults. In these animals, nearby support cells act as hair cell progenitors. Differences in support cell shape, structure, and motility have been observed between regenerating and non-regenerating organisms, but whether these factors directly regulate regenerative capacity is unclear. This project seeks to use live imaging of zebrafish lateral line neuromasts to characterize support cell shape and dynamics during hair cell death, differentiation, and regeneration. One aim of the project is to use quantitative cell shape analysis to determine the relationship between support cell shape and fate. Ultimately, this may make it possible to use cell morphology to predict which support cells will act as hair cell progenitors. Another focus of the project is to understand how actomyosin contractility regulates hair cell extrusion, differentiation, and regeneration. The actin cytoskeleton is a major determinant of cell shape and dynamics, and support cell F-actin structure is known to differ in adult mammals compared to non-mammals. The information gained from these studies may help investigators design therapies to stimulate hair cell regeneration in adult mammals. It will be important to consider how regenerative therapies will impact the shape and structure of cells in the inner ear because the function of inner ear organs is highly dependent on correct cell orientation and organization. This project will take place in Dr. David Raible's lab at the University of Washington, a rich training environment with abundant expertise and resources to study zebrafish hair cells. These studies will be done in collaboration with experts in quantitative cell biology and biophysics, who will provide additional resources and guidance for the principal investigator. The project includes a training plan that will see the principal investigator gain imaging, modeling, and programming experience to become an independent research scientist applying interdisciplinary approaches to cell and developmental biology.